Human Combinatorial Antibody Library (HuCAL®) technology is a unique and innovative concept for in vitro generation of highly-specific and completely human antibodies - for a schematic drawing of available antibody formats see "Pictures" below.

Please Note:HuCAL® antibodies are sold under license of U.S. Patents 6,300,064; 6,696,248; 6,708,484;6,753,136; and European Patent 0,859,841 and corresponding patents.

Use of HuCAL® antibodies in ELISAELISA can be performed using HuCAL® antibodies in protocols used for other poly- or monoclonal antibodies, provided a suitable secondary antibody is used. As HuCAL® antibodies do not contain the Fc domain, we recommend using an anti-human Fab secondary antibody (use of polyclonal antiserum amplifies the signal). Monoclonal antibodies against the tag (e.g. anti-Myc, anti-His-6) can also be used. The bivalent format of the HuCAL® mini-antibodies (Fab-dHLX or Fab-A) is recommended for ELISA assays with immobilized antigen because their avidity is higher, similar to that of full IgGs.(A) Indirect ELISAIndirect ELISA is recommended as a control assay to test the performance of reagents. In indirect ELISA, the antigen is immobilized on a surface, such as a well of a microtiter plate. The plate is blocked to prevent non-specific binding of antibodies, and a specific antibody is added to each well. The plate is washed to remove unbound antibody, leaving only the specific antigen-antibody complexes of interest. A secondary antibody is added. This is conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase, and binds the antibody-antigen complexes. The plate is washed and the appropriate substrate is applied. The resulting chromogenic or fluorescent signal is viewed using a spectrophotometer.ProtocolIndirect ELISA with fluorescence readout1. Coat the antigen by adding 20 µl of a solution of 5 μg/ml antigen in PBS to several wells of a 384-well Maxisorp(TM) microtiter plate, and incubate overnight at 4°C.Use a black, 384-well ELISA microtiter plate: square well, flat bottom MaxiSorp(TM) PS (NUNC, Cat. No. 460518).2. Wash the microtiter plate twice with PBST.3. Block the microtiter plate by adding 100 µl of 5% non-fat dried milk in PBST to each well, and incubate for 1–2 hours at room temperature.4. Wash the microtiter plate twice with PBST.5. Transfer 20 µl of HuCAL® antibody to each well. We recommend a standard concentration of 2 µg/ml in PBST or HiSpec buffer BUF049). Incubate for 1 hour at room temperature.Titrate using different concentrations of HuCAL® antibody.6. Wash the microtiter plate 5 times with PBST.7. Add 20 µl of secondary antibody to each well and incubate for 1 hour at room temperature.We recommend using anti-human Fab:AP .8. Wash the microtiter plate 5 times with PBST.9. Add 20 µl of AttoPhos® (Roche) to each well and measure fluorescence after 10 minutes (excitation: 440±25 nm, emission: 550±35 nm).Note: Instead of the 384-well format, a 96-well plate can be used (e.g. black, flat bottom MaxiSorp(TM)PS, NUNC, Cat. No. 437111). For the 96-well format use 100 µl instead of 20 µl of antigen, antibodies or substrate and 300 μl for the blocking step. Instead of an alkaline phosphatase-conjugated secondary antibody, anHRP-conjugated anti-Fab can be used in combination with QuantaBlu® reagent (Pierce Cat. No. 15169, excitation: 320±25 nm, emission: 430±35 nm).If detection is done with a chromagenic substrate (e.g. BM Blue Soluble Peroxide Substrate, Roche, Cat. No. 1484281), transparent plates must be used.(B) Direct ELISAIn direct ELISA, the primary antibody is labeled, for example, by genetic fusion with alkaline phosphatase. This avoids use of a secondary antibody, saving time and money, which is valuable for assays that are frequently run. However, the system lacks the amplification effect generated by the secondary antibody in indirect ELISA and is therefore less sensitive.ProtocolDirect ELISA with alkaline phosphatase-conjugated primary antibody and fluorescence readout1. Coat the antigen by adding 20 µl of a solution of 5 µg/ml antigen in PBS to several wells of a 384-well Maxisorp(TM) microtiter plate, and incubate overnight at 4°C.Use a black, 384-well ELISA microtiter plate: square well, flat bottom, MaxiSorp(TM) PS (NUNC, Cat. No. 460518).2. Wash the microtiter plate twice with PBST.3. Block the microtiter plate by adding 100 µl of 5% non-fat dried milk in PBST to each well, and incubate for 1-2 hours at room temperature.4. Wash the microtiter plate twice with PBST.5. Transfer 20 µl of labeled HuCAL® antibody to each well. We recommend a standard concentration of 2 µg/ml in PBST. Incubate for 1 hour at room temperature. Titrate using different concentrations of HuCAL® antibody.6. Wash the microtiter plate 5 times with PBST.7. Add 20 µl of AttoPhos® (Roche) to each well and measure the fluorescence after 10 minutes (excitation: 440±25 nm, emission: 550±35 nm).(C) Sandwich ELISASandwich ELISA is a very sensitive and specific method of detecting antigens, and provides fast and accurate determination of the concentration of antigen in a sample. If a purified antigen standard is available, the method can also measure the absolute concentration of antigen in a given sample. The technique uses two antibodies, both of which are specific to the antigen of interest, and which bind the antigen at non-overlapping epitopes. In sandwich ELISA, it is possible to use a HuCAL® antibody as the capture or detection antibody together with an existing commercial antibody, or to use two different HuCAL® antibodies as the sandwich pair. The bivalent format of HuCAL® mini-antibodies is highly suitable for both capture and detection. When two HuCAL® antibodies are used, detection is best performed using a labeled anti-tag tertiary antibody (e.g. Fab-A-FH HuCAL® antibody with HRP-conjugated rabbit anti-bacterial alkaline phosphatase). Alternatively, the detection antibody can be biotinylated and visualized with streptavidin-AP.ProtocolsSandwich ELISA using two HuCAL® antibodies1. Coat the capture antibody by adding 20 µl of a solution of 5 µg/ml in PBS to several wells of a 384-well Maxisorp(TM) microtiter plate, and incubate overnight at 4°C.Use a black, 384-well ELISA microtiter plate: square well, flat bottom, MaxiSorp(TM) PS (NUNC, Cat. No. 460518).2. Wash the microtiter plate twice with PBST.3. Block the microtiter plate by adding 100 µl of 5% BSA in PBST to eachwell, and incubate for 1 hour at room temperature.4. Wash the microtiter plate twice with PBST.5. Add 20 µl of antigen to each well of the microtiter plate, and incubate for 1 hour at room temperature.Use a concentration range of the antigen in PBS.6. Wash the microtiter plate 5 times with PBST.7. Add 20 µl of HuCAL® detection antibody in Fab-A-FH format (2 µg/ml concentration in PBST or HiSpec buffer) to each well, and incubate for 1 hour at room temperature.8. Wash the microtiter plate 5 times with PBST.9. Transfer 20 µl of HRP-conjugated rabbit anti-bacterial alkaline phosphatase antibody and incubate for 1 hour at room temperature.Use rabbit anti-bacterial alkaline phosphatase.10. Wash the microtiter plate 5 times with PBST.Add 20 µl of QuantaBlu® (Pierce) to each well and measure the fluorescence directly (excitation: 320±25 nm, emission: 420±35 nm).Sandwich ELISA with polyclonal mouse and HuCAL® antibody1. Coat the HuCAL® capture antibody by adding 20 µl of a solution of 5 µg/ml in PBS to several wells of a 384-well Maxisorp(TM) microtiter plate, and incubate overnight at 4°C.2. Wash the microtiter plate twice with PBST.3. Block the microtiter plate by adding 100 µl of 5% BSA in PBST to each well, and incubate for 1 hour at room temperature.4. Wash the microtiter plate twice with PBST.5. Add 20 µl of antigen to each well of the microtiter plate, and incubate for 1 hour at room temperature.Use a concentration range of the antigen in PBS .6. Wash the microtiter plate 5 times with PBST.7. Add 20 µl of biotinylated polyclonal detection antibody (concentration as specified by the manufacturer) in HiSpec buffer to each well, and incubate for 1 hour at room temperature.8. Wash the microtiter plate 5 times with PBST.9.Transfer 20 µl alkaline phosphatase conjugated streptavidin and incubate for 1 hourat room temperature.(D) Competition (or inhibition) ELISACompetition (or inhibition) ELISA is ideal when only one suitable antibody is available for the target of interest or when the antigen is too small to be detected e.g. a hapten. The technique measures the concentration of a substance by its ability to interfere with an established pre-titrated system. The primary antibody is first incubated with the free antigen. It is then added to an antigen-coated well, and the plate is washed to remove unbound antibody. The amount of antibody that binds the immobilized antigen is detected using a secondary antibody linked to a detection system such as horseradish peroxidase. The appropriate substrate is applied and the resulting chromogenic or fluorescent signal is viewed using a spectrophotometer. The higher the concentration of free antigen in the sample, the less antibody is available to bind the immobilized antigen, and, therefore, the weaker the signal; conversely, lower amounts of free antigen in solution generate stronger signals. Other variations of competition ELISA are of course possible.Protocol1. Coat the antigen by adding 50 µl of a solution of 5 µg/ml antigen in PBS to several wells of a 384-well Maxisorp(TM) microtiter plate, and incubate overnight at 4°C.2. Wash the microtiter plate twice with PBST.3. Block the microtiter plate by adding 100 µl of 5% non-fat dried milk in PBST to each well, and incubate for 1-2 hours at room temperature.4. Incubate 25 µl of antigen solution with 25 µl of HuCAL® antibody (final concentration 2 µg/ml) for 1 hour at room temperature. Use a range of concentrations of the antigen in solution, in PBS5. Wash the microtiter plate twice with PBST.6. Transfer 50 µl of the HuCAL® antibody/antigen mix to each well of the microtiter plate, and incubate for 1 hour at room temperature.7. Wash the microtiter plate 5 times with PBST.8. Transfer 50 µl of secondary antibody (anti-human Fab:AP and incubate for 1 hour at room temperature.9. Wash the microtiter plate 5 times with PBST.10. Add 20 µl of AttoPhos® (Roche) to each well, incubate for 10 minutes at room temperature, and measure fluorescence (excitation: 440±25nm, emission: 550±35nm).

Storage

Prior to reconstitution store at 2-8°C.Following reconstitution store the antibody undiluted at 2-8°C for one monthor (in aliquots) at -20°C for longer.Avoid repeated freezing and thawing.Shelf life: one year from despatch.

Lyophilised bivalent human recombinant Fab selected from the HuCAL® GOLD phage display library. Expressed in E. coli and purified using metal chelate affinity chromatography. This Fab fragment is dimerized via a helix-turn-helix motif.The antibody is tagged with a myc-tag (EQKLISEEDL) and a his-tag (HHHHHH) at the C-terminus of the antibody heavy chainPurified

Reconstitution:

Reconstitute with 1.0 ml distilled water (AM05849PU-N) or 0.5ml (AM05849PU-S).

Specificity

Specificity:

The HuCAL® antibody specifically recognises the idiotypic determinants expressed by the Rituximab humanised monoclonal antibody.Rituximab is a therapeutic reagent directed against the human CD20 cell surface antigen.It does not recognise other CD20 antibodies.

Accessory Products

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